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WO2022202785A1 - Peptide, et agent de fusion cellulaire et composition pharmaceutique pour cancérothérapie contenant ledit peptide - Google Patents

Peptide, et agent de fusion cellulaire et composition pharmaceutique pour cancérothérapie contenant ledit peptide Download PDF

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Publication number
WO2022202785A1
WO2022202785A1 PCT/JP2022/013074 JP2022013074W WO2022202785A1 WO 2022202785 A1 WO2022202785 A1 WO 2022202785A1 JP 2022013074 W JP2022013074 W JP 2022013074W WO 2022202785 A1 WO2022202785 A1 WO 2022202785A1
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amino acid
polypeptide
seq
group
acid sequence
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美智子 甲賀
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a polypeptide, a cell fusion agent containing the same, and a pharmaceutical composition for treating cancer. According to the present invention, cells can be efficiently fused.
  • Non-Patent Documents 1 and 2 Cell fusion is a phenomenon that was discovered because Sendai virus has the effect of fusing cells. Currently, cell fusion is used for breed improvement, production of monoclonal antibodies, and the like. In addition to using viruses, it is known that cell fusion also occurs by the protoplast-PEG method or electrical stimulation.
  • the present inventors considered whether there is a method for efficient cell fusion. We also thought that cancer cells could be killed by cell fusion. Accordingly, it is an object of the present invention to provide a method for efficient cell fusion. Another object of the present invention is to provide a method for killing cancer cells by cell fusion or a method for fusing virus envelopes by membrane fusion.
  • the present invention provides [1] (1) The following formula (I): -ZX m -Y (I) (wherein, Z is a hydrophilic linker, X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine, and Y is a carboxyl group or an amino group, m is an integer of 1 to 5, and when m is 2 to 5, the hydrophilic amino acid residues may be the same amino acid residue or a combination of different amino acid residues).
  • a polypeptide comprising an amino acid sequence having a group represented by formula (I) at the N-terminus and having cell fusion activity, [2] The polypeptide of [1], wherein the amino acid sequences represented by SEQ ID NOs: 1 to 8 have a methyl group at the N-terminus; [3] The polypeptide of [1] or [2], wherein Z is -NH-(CH 2 CH 2 O) n -CO-, and n is an integer of 1 to 4; [4] an antibody or an antigen-binding fragment thereof that binds to the polypeptide of any one of [1] to [3]; [5] a cell fusion agent containing the polypeptide of any one of [1] to [3] as an active ingredient; [6] A pharmaceutical composition comprising the polypeptide according to any one of [1] to [3]
  • hydrophilic polypeptide of the present invention cells can be efficiently fused.
  • Hydrophilic polypeptides of the invention are capable of fusing the viral envelope.
  • the hydrophilic polypeptide of the present invention can be used as an active ingredient of a pharmaceutical composition for treating cancer.
  • Fig. 10 is a micrograph (x 100) when peptide 12 was allowed to act on RFL cells (A) and RM4 cells (B).
  • Fig. 10 is a micrograph (x100) when peptide 13 was allowed to act on RFL cells (A) and RM4 cells (B).
  • Fig. 3 is micrographs (x 100) when peptide 15 was allowed to act on RFL cells (A) and RM4 cells (B).
  • FIG. Fig. 2 shows micrographs (x100) of the reaction of peptide 16 on RFL cells (A) and RM4 cells (B). It is a micrograph (x100) when peptide 17 was allowed to act on RFL cells (A) and RM4 cells (B).
  • Fig. 2 is micrographs (x 100) when peptide 18 was allowed to act on RFL cells (A) and RM4 cells (B).
  • 1 is an electron micrograph of RFL cells treated with peptide 16.
  • FIG. Fig. 3 is a graph showing the induction of apoptosis measured by the activation of caspase-3/7 when peptide 16 was applied to RFL cells.
  • FIG. 3 is a graph showing the induction of apoptosis measured by the activation of caspase-3/7 when peptide 16 was applied to RM4 cells.
  • 1 is an electron micrograph of HVJ virus (Sendai virus) reacted with peptide 16.
  • polypeptide of the present invention has the following formula (I) at the N-terminus of an amino acid sequence selected from the group consisting of (1) amino acid sequences represented by SEQ ID NOs: 1 to 8: -ZX m -Y (I) wherein Z is a hydrophilic linker and X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine.
  • Z is a hydrophilic linker
  • X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine.
  • Y is a carboxyl group or an amino group
  • m is an integer of 1 to 5
  • the hydrophilic amino acid residues may be the same amino acid residue, or a combination of different amino acid residues (may be acceptable). That is, the polypeptide of the present invention has hydrophilic amino acids bound via a hydrophilic linker. Hydrophilic amino acid attachment improves the solubility of the polypeptide in aqueous solutions, allowing efficient cell fusion.
  • the amino acid sequences represented by SEQ ID NOs: 1 to 8 are as follows.
  • Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala SEQ ID NO: 1
  • Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala SEQ ID NO: 2
  • Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala SEQ ID NO: 3
  • Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala SEQ ID NO: 4
  • Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala SEQ ID NO: 5
  • Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala SEQ ID NO: 6
  • Pro-Ile-Val-Ser-Gln-Thr-Thr-Thr-Ala-Ile-Ala SEQ ID NO: 7
  • the C-terminal alanine (Ala) of the amino acid sequences represented by SEQ ID NOs: 1 to 8 and the group represented by the above formula (I) are linked by a peptide bond (--CO--NH--).
  • an amino acid residue means the OH of the carboxyl group (COOH) of an amino acid and the H of the amino group (NH 2 ) removed.
  • Z in the group of formula (I) is a hydrophilic linker that connects the amino acid sequences represented by SEQ ID NOs: 1 to 8 and hydrophilic amino acid residues.
  • the polypeptides having the amino acid sequences represented by SEQ ID NOs: 1 to 8 exhibit the effects of the present invention, such as cell fusion activity, without being affected by the hydrophilic amino acid residues. be able to.
  • the hydrophilic linker Z is not particularly limited and includes, for example, a hydrocarbon group containing a heteroatom.
  • miniPEG "-NH-(CH 2 CH 2 O) n -CO-" is used. be able to.
  • Said n is, for example, 1 to 4, preferably 2 to 3, and more preferably 2.
  • Said hydrophilic amino acid residue is selected from serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine or cysteine, preferably arginine, histidine or lysine.
  • the number of amino acid residues m is 1-5, preferably 2-4.
  • the hydrophilic amino acid residue may be an L-amino acid residue or a D-amino acid residue, but is preferably a D-amino acid from the viewpoint of suppressing in vivo degradation.
  • the amino acid residue may be the same amino acid residue or a combination of two or more amino acid residues. Specific amino acid residue sequences include D-Lys-D-Lys-D-Lys, D-Arg-D-Arg-D-Arg, or D-His-D-His-D-His.
  • polypeptide of the present invention is (2) in the amino acid sequences represented by SEQ ID NOS: 1 to 8, at the N-terminus of the amino acid sequences in which 1 to 4 amino acids are deleted, substituted, inserted, and / or added. It contains an amino acid sequence having a group represented by formula (I) and has cell fusion activity.
  • amino acid sequences represented by SEQ ID NOS: 1 to 8 1 to 4 amino acids are deleted, substituted, inserted, and/or added to the N-terminus of the amino acid sequence represented by the formula (I) (hereinafter sometimes referred to as "functionally equivalent variant") comprising an amino acid sequence having a consists of an amino acid sequence having a group represented by the formula (I) at the N-terminus of an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, and/or added, and has cell fusion activity including polypeptides.
  • the polypeptide of the present invention can exhibit cell fusion activity by a polypeptide consisting of the 10 amino acid sequences such as the amino acid sequences represented by SEQ ID NOs: 1 to 8.
  • the group of formula (I), other amino acids, polypeptides, or proteins bind to the polypeptide of the present invention as long as the specific structure exhibiting cell fusion activity in the 10 amino acid sequence is not destroyed.
  • the polypeptides of the present invention can exhibit cell fusion activity.
  • Functionally equivalent variants have 1 to 4, preferably 1 to 3, more preferably 1 to 2, most preferably 1 at one or more positions in the amino acid sequences represented by SEQ ID NOs: 1 to 8.
  • a polypeptide comprising an amino acid sequence having a group represented by the above formula (I) at the N-terminus of an amino acid sequence in which 10 amino acids have been deleted, substituted, inserted, and/or added, and having cell fusion activity As long as it is not particularly limited.
  • a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5 is a functionally equivalent variant in which one amino acid is substituted, and SEQ ID NO: A polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 is a functionally equivalent variant in which two amino acids are substituted, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 has three amino acids. is a functionally equivalent variant in which is substituted, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 is a functionally equivalent variant in which 4 amino acids are substituted.
  • “Deletion, substitution, insertion and/or addition of 1 to 4 amino acids” in functionally equivalent variants are conservative substitutions that maintain the function of the polypeptide of the present invention.
  • a “conservative substitution” can be performed, for example, without limitation, by replacing an amino acid residue with another, chemically similar amino acid residue. For example, replacing one hydrophobic residue with another hydrophobic residue, replacing one polar residue with another polar residue having the same charge, and the like.
  • Functionally similar amino acids for which such substitutions can be made are known in the art for each amino acid.
  • Nonpolar (hydrophobic) amino acids include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like.
  • Polar (neutral) amino acids include, for example, glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like.
  • positively charged (basic) amino acids include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • the N-terminal proline is preferably methylated. That is, the N-terminal proline is preferably methylated proline. Although it is not limited, methylation of proline at the N-terminus can exhibit even better cell fusion activity.
  • the polypeptide of the present invention is preferably produced by a chemical synthesis method.
  • the polypeptide of the present invention has cell fusion activity.
  • Cell fusion by the polypeptide of the present invention fuses several cells to form a fused cell with multiple nuclei.
  • the fused cells are induced to undergo apoptosis after cell fusion, resulting in cell death.
  • Antibodies eg, polyclonal antibodies or monoclonal antibodies
  • Antibodies that react with the polypeptide of the present invention can be obtained by directly administering the polypeptide of the present invention or fragments thereof to various animals.
  • DNA vaccine methods Rost, E. et al., Proc. Natl. Acad. Sci. USA, 91, 9519-9523, 1994; or Donnelly , J. J. et al., J. Infect. Dis., 173, 314-320, 1996).
  • Polyclonal antibodies are obtained by, for example, animals sensitized by intraperitoneally, subcutaneously, or intravenously with an emulsion of the polypeptide of the present invention or a fragment thereof in an appropriate adjuvant (e.g., Freund's complete adjuvant). (eg, rabbit, rat, goat, chicken, etc.) serum or eggs.
  • adjuvant e.g., Freund's complete adjuvant
  • a polyclonal antibody can be separated and purified from serum or eggs thus produced by a conventional protein isolation and purification method. Examples of such separation and purification methods include centrifugation, dialysis, salting-out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, or the like.
  • Monoclonal antibodies can be easily produced by those skilled in the art, for example, by the cell fusion method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975). That is, an emulsion prepared by emulsifying the polypeptide of the present invention or a fragment thereof in an appropriate adjuvant (e.g., Freund's complete adjuvant) is repeatedly inoculated into mice peritoneally, subcutaneously, or intravenously several times every few weeks. to immunize. After the final immunization, spleen cells are removed and fused with myeloma cells to produce hybridomas.
  • an adjuvant e.g., Freund's complete adjuvant
  • myeloma cells for obtaining hybridomas for example, myeloma cells having markers such as hypoxanthine-guanine-phosphoribosyltransferase deficiency or thymidine kinase deficiency (for example, mouse myeloma cell line P3X63Ag8.U1) can be used. .
  • polyethylene glycol for example, can be used as a fusing agent.
  • a medium for hybridoma production for example, 10 to 30% fetal bovine serum is added to a commonly used medium such as Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, or RPMI-1640. can be used in addition.
  • Fusion strains can be selected by the HAT selection method.
  • Hybridoma screening can be carried out using culture supernatants by well-known methods such as ELISA or immunohistochemical staining to select clones of hybridomas secreting the antibody of interest.
  • the monoclonality of hybridomas can be ensured by repeating subcloning by the limiting dilution method.
  • the hybridomas thus obtained are cultured in culture medium for 2-4 days or intraperitoneally in pristane-pretreated BALB/c mice for 10-20 days to produce purifiable amounts of antibody. can do.
  • the monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by a conventional polypeptide isolation and purification method.
  • separation and purification methods include centrifugation, dialysis, salting-out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, or the like.
  • a monoclonal antibody or an antibody fragment containing a portion thereof can be obtained by inserting all or part of the gene encoding the monoclonal antibody into an expression vector and introducing the gene into an appropriate host cell (e.g., E. coli, yeast, or animal cells). It can also be produced.
  • Antibodies (including polyclonal antibodies and monoclonal antibodies) separated and purified as described above are digested with a polypeptide-degrading enzyme (e.g., pepsin or papain) in a conventional manner, followed by isolation of the polypeptide in a conventional manner.
  • a polypeptide-degrading enzyme e.g., pepsin or papain
  • an antigen-binding fragment containing an active antibody portion such as F(ab) 2 , Fab, Fab, or Fv, can be obtained.
  • an antibody that reacts with the polypeptide of the present invention can be detected by the method of Clackson et al. or the method of Zebedee et al. USA, 89, 3175-3179, 1992) as single chain Fv or ab.
  • Human antibodies can also be obtained by immunizing transgenic mice in which mouse antibody genes have been replaced with human antibody genes (Lonberg, N. et al., Nature, 368, 856-859, 1994).
  • the cell fusion agent of the present invention contains the polypeptide of the present invention as an active ingredient.
  • the cell fusion agent of the present invention may contain one polypeptide alone, or may contain two or more polypeptides in combination.
  • the content of the polypeptide in the cell fusion agent is not particularly limited, but is, for example, 0.1 to 100% by weight, preferably 10 to 100% by weight, more preferably 30 to 90% by weight. is.
  • the cell fusion agent of the present invention may contain carriers (e.g., water or buffers), excipients, diluents, preservatives, stabilizers, preservatives, antioxidants, etc., as components other than polypeptides. good.
  • the cell fusion agent of the present invention can be used for plant breeding, monoclonal antibody production, and the like. According to the cell fusion agent of the present invention, cells can be fused efficiently.
  • Cells to be fused by the cell fusion agent of the present invention are not particularly limited, and include microbial cells, plant cells, and animal cells.
  • Animal cells include nucleated cells (e.g., blood cells, lymphoid cells, cells constituting internal organs) of vertebrates (e.g., mammals) such as mice, rats, rabbits, guinea pigs, goats, sheep, horses, and cows, and mammals. Examples include cancer cells derived from animals.
  • the temperature for cell fusion is not particularly limited as long as cell fusion occurs, but is, for example, 0 to 40°C, preferably 10 to 38°C.
  • the treatment time is not particularly limited, it is preferably 1 minute to 2 hours.
  • the pharmaceutical composition of the present invention contains the polypeptide of the present invention as an active ingredient.
  • Diseases that can be prevented or treated by the pharmaceutical composition of the present invention are not particularly limited.
  • cancer cells can be fused, cancer cells can be killed, and cancer can be treated.
  • the peptides of the present invention can induce apoptosis in cells by cell fusion. Caspase-3/7 or Annexin V is activated in the fused cells, and apoptosis is induced. Cancer cells can be killed by inducing apoptosis in the fused cells.
  • Cancers that can be treated with the pharmaceutical composition of the present invention include tongue cancer, gingival cancer, malignant lymphoma, malignant melanoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer, glioma, meningioma, and nerve cancer.
  • glioma neuroblastoma, papillary adenocarcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, primary lung cancer, squamous cell carcinoma, adenocarcinoma, alveolar carcinoma, large cell undifferentiated carcinoma, small cell undifferentiated carcinoma Cancer, carcinoid, testicular tumor, prostate cancer, breast cancer, Paget's disease of the breast, breast sarcoma, bone tumor, thyroid cancer, gastric cancer, liver cancer, acute myelogenous leukemia, acute promyelogenous leukemia, acute myelomonocytic leukemia, acute monocytic leukemia Cellular leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma, multiple myeloma, primary macroglobulinemia, childhood leukemia, Esophageal cancer,
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples include powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills. and parenteral agents such as injections, external solutions, ointments, suppositories, creams for topical administration, and eye drops.
  • Oral agents include gelatin, sodium alginate, starch, cornstarch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, and magnesium stearate.
  • polyethylene glycol such as physiological saline or Ringer's solution
  • water-insoluble solvents such as vegetable oils or fatty acid esters
  • isotonic agents such as glucose or sodium chloride, and solubilizers.
  • stabilizers, preservatives, suspending agents, emulsifying agents and the like can optionally be used.
  • the dosage when using a pharmaceutical composition can be appropriately determined according to, for example, the type of active ingredient to be used, the type of disease, the patient's age, sex, weight, degree of birth, or administration method, It can be administered orally or parenterally.
  • the oral intake of the pharmaceutical composition of the present invention is preferably 0.01 to 100 mg/kg of polypeptide per day for adults.
  • the above administration method is an example, and other administration methods may be used.
  • the administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans are desirably determined by controlled clinical trials.
  • the mode of administration is not limited to pharmaceuticals, and can be given in various forms such as functional foods, health foods (including beverages), or food and drink as feed.
  • a pharmaceutical composition containing a polypeptide can be produced using a known method for producing pharmaceuticals, except that the polypeptide is included as an active ingredient.
  • the pharmaceutical composition of the present invention can contain other ingredients.
  • the other components include edible oils and fats, water, glycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, glycerin organic acid fatty acid ester, polyglycerin fatty acid ester, calcium stearoyl lactylate, sodium stearoyl lactylate, Emulsifiers such as polyoxyethylene sorbitan fatty acid esters, thickening stabilizers such as locust bean gum, carrageenan, alginic acids, pectin, xanthan gum, crystalline cellulose, carboxymethyl cellulose, methyl cellulose, agar, glucomannan, gelatin, starch, or modified starch, salt or potassium chloride, acidulants such as acetic acid, lactic acid, or gluconic acid; sugars or sugar alcohols; sweeteners such as stevia or aspartame; coloring
  • the total content of these other ingredients in the pharmaceutical composition of the present invention is preferably 8% by mass or less, more preferably 40% by mass or less, and still more preferably 20% by mass or less.
  • the pharmaceutical composition of the present invention can be administered to humans, but the subject of administration may be animals other than humans, such as pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; Domestic animals such as pigs; experimental animals such as mice and rats; and animals raised in zoos and the like.
  • the cancer treatment method of the present invention comprises the step of administering an effective amount of the polypeptide to a subject in need of treatment. That is, the polypeptides of the present invention can be used in cancer treatment methods. Cancer can be treated by administering an effective amount of the pharmaceutical composition to humans or animals.
  • polypeptides for treating cancer are for the treatment of cancer.
  • Said polypeptides can be used in methods of treating cancer.
  • disclosed herein are polypeptides that are for the treatment of cancer.
  • Said polypeptides can be used in the manufacture of pharmaceutical compositions.
  • the present specification discloses the use of polypeptides for the manufacture of pharmaceutical compositions.
  • Said pharmaceutical composition is, but is not limited to, a pharmaceutical composition for treating cancer.
  • the antiviral agent of the present invention contains, as an active ingredient, (A) the polypeptide of the present invention, or (B) (b1) an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8.
  • Polypeptide (b2) a polypeptide comprising an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, and/or added in the amino acid sequences represented by SEQ ID NOS: 1 to 8, and having cell fusion activity a peptide, (b3) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8 and having a methyl group at the N-terminus, or (b4) SEQ ID NO: 1 A polypeptide comprising an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence represented by 8 and has cell fusion activity, wherein the N-terminal methyl A polypeptide having a group. Said polypeptides are capable of fusing viral envelopes and exhibit antiviral activity. That is, it can be used as an antiviral agent for enveloped viruses.
  • enveloped viruses include, but are not limited to, poxviridae, baculoviridae, rhabdoviridae, bunyaviridae, togaviridae, herpesviridae, paramyxoviridae, orthomyxoviridae, retroviruses, and retroviruses.
  • influenza viruses of the family Viridae, Arenaviridae, or Coronaviridae, and more specifically influenza viruses such as avian influenza virus, human influenza virus, swine influenza virus, hepatitis B virus, hepatitis C virus, Human immunodeficiency virus, varicella-zoster virus, herpes simplex virus, human herpes virus, mumps virus, respiratory syncytial virus, Ebola virus, rubella virus, coronavirus, measles virus, arbovirus, SARS virus, hepatitis A virus, hepatitis D viruses, hepatitis E virus, yellow fever virus, adult T-cell leukemia virus, rabies virus, hantavirus, dengue virus, nipah virus, or lyssa virus.
  • influenza viruses such as avian influenza virus, human influenza virus, swine influenza virus, hepatitis B virus, hepatitis C virus, Human immunodeficiency virus, varicella-
  • the polypeptide can be used in a method for treating the viral disease.
  • Said polypeptides can be used as polypeptides for the treatment of viral diseases.
  • Said polypeptide can be used for the manufacture of a pharmaceutical composition for treating viruses.
  • the 2nd and 9th leucines or isoleucines exhibit cell fusion activity even when substituted with each other, so the 2nd and 9th amino acids can be substituted, and by substitution with other amino acids (e.g., valine) It is also considered that there is a high possibility that it also exhibits cell fusion activity.
  • 5th and 6th threonine and glutamine also show cell fusion activity even if they are replaced with each other. , it is highly likely that substitution with other amino acids with similar properties will also exhibit cell fusion activity.
  • alanine at positions 8 and 10 may exhibit cell fusion activity even when substituted with amino acids with similar properties, such as glycine.
  • proline methylation at the N-terminus is not essential for the cell fusion ability of each peptide and induction of cancer cell apoptosis. Therefore, peptides in which one or more amino acids are added to the N-terminal proline can also exhibit cell fusion and apoptosis-inducing ability.
  • the mechanism by which the polypeptide of the present invention has an anticancer effect has not been completely elucidated, it is presumed as follows. However, the present invention is not limited by the following assumptions. The polypeptides of the present invention are presumed to be able to fuse cancer cells and induce apoptosis in the cells, thereby killing the cancer cells. Moreover, the cell fusion is induced regardless of the type of cancer.
  • the polypeptides of the present invention are believed to be effective against many types of cancer. Furthermore, the reason why the polypeptide of the present invention exhibits excellent water solubility is that it has the group represented by the formula (I) at the C-terminus of the amino acid sequence represented by the SEQ ID NOs: 1 to 8. Conceivable.
  • the amino acid sequences represented by SEQ ID NOs: 1 to 8 contain many hydrophobic amino acids. Therefore, it is thought that several hydrophilic amino acids are bound via hydrophilic linkers to exhibit excellent water solubility as a whole. be done.
  • Example 1>> the N-terminal proline of the amino acid sequences represented by SEQ ID NOS: 1 to 8 below was methylated, and the peptide of the amino acid sequence represented by SEQ ID NO: 1 (proline is not methylated). , and N-terminal proline of the amino acid sequence represented by SEQ ID NO: 9 was synthesized. Peptide synthesis was outsourced to Greiner/Fasmax.
  • the amino acid sequence represented by SEQ ID NO:9 is an amino acid sequence obtained by adding threonine and alanine to the C-terminal side of the amino acid sequence represented by SEQ ID NO:1.
  • peptide 1 CH3 -Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala
  • peptide 2 CH3 -Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala
  • peptide 3 CH3 -Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala
  • peptide 4 CH3 -Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala
  • peptide 5 CH3 -Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala
  • Fmoc amino acids were activated with HBTU/HOBT solution (HBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniu Hexafluorophosphate; HOBT: 1-Hydroxybenzotriazole), followed by DIEA ( N,N'-Diisopropylethylamine) was added to condense the amino acids.
  • HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluroniu Hexafluorophosphate
  • HOBT 1-Hydroxybenzotriazole
  • DIEA N,N'-Diisopropylethylamine
  • TFA trifluoroacetic acid
  • 0.25 mL H 2 O 0.375 g phenol, 0.125 mL ethanedithiol and 0.25 mL thioanisole
  • the resulting crude peptide was purified using RP-HPLC and lyophilized. Purity of purification was examined by HPLC and MS under the following conditions.
  • a polypeptide was synthesized in which miniPEG-D-Lys-D-Lys-D-Lys-NH was bound to the N-terminus of the amino acid sequences represented by SEQ ID NOs: 1 to 8 above.
  • Peptide synthesis was outsourced to Peptide Institute Co., Ltd.
  • the crude peptide was solidified with ether (50 mL), purified and lyophilized on a reverse-phase HPLC ODS column (YMC-Pack ODS-A ⁇ 30 ⁇ 250 mm) using water containing 0.1% trifluoroacetic acid and acetonitrile as eluents. , 180 mg of the trifluoroacetate peptide was obtained. Thereafter, the resulting trifluoroacetate peptide (180 mg) was salt-exchanged with an ion exchange column (DOWEX 1x2 100-200 Mesh Anion Exchange Resin CH3COO form) using 5% acetic acid as an eluent, and lyophilized to obtain the desired product. 120 mg of product was obtained as a white lyophilized powder of the acetate salt. Other peptides were also produced according to the procedure for producing peptide 16 described above.
  • the peptides 11 to 18 were allowed to act on RFL cells (rat fetal lung-derived cells) or RM4 cells to examine the cell fusion activity of the peptides.
  • RFL cells or RM4 cells (2 ⁇ 10 6 cells) were suspended in 6 mL of RPMI-1640 (Wako, 189-02025) medium supplemented with 5% FBS (Biosera, Cat No. 015BS493), and plated on a 24-well plate (Iwaki, 2820- 024), 8 ⁇ 10 4 cells/0.25 mL were dispensed into each well and cultured.
  • FIG. 1-8 show micrographs of RFL cells (A) or RM4 cells (B). In RFL cells or RM4 cells, cells were fused and fused cells with multiple nuclei were seen. In addition, FIG. 9 shows electron micrographs of peptide 16 acting on RFL cells.
  • Example 4>> the apoptotic potential of peptide 16 was examined using RFL cells and RM4 cells.
  • Caspase-3/7 activity which is an apoptosis index, was measured using the IncuCyte S3 Viability Analysis System (Essen Biosciences).
  • Caspase-3/7 activity is measured using an inert, non-fluorescent (DEVD) substrate that can cross cell membranes. Cleavage of the substrate by activated Caspase-3/7 releases a DNA-bound green fluorescent label, and the intensity of the green fluorescence measures the activity of Caspase-3/7.
  • DEVD inert, non-fluorescent
  • FIG. 10 shows caspase-3/7 activity in RFL cells
  • FIG. 11 shows caspase-3/7 activity in RM4 cells. In both RFL cells and RM4 cells, the activity increased sharply between 10 and 20 hours and gradually increased thereafter. On the other hand, in RFL cells and RM4 cells not treated with peptide 16, the caspase-3/7 activity was not increased.
  • peptide 16 was allowed to act on HVJ virus (Sendai virus), and the ability to fuse with the virus was examined.
  • FIG. 12 shows an electron micrograph. Fusion of the HVJ virus envelope was observed.
  • A549 cells human alveolar epithelial adenocarcinoma cells
  • Peptide 16 was administered intravenously through the tail vein at a dose of 18.75 mg/kg or 37.5 mg/kg (Peptide 16 group-1 or Peptide 16 Group-2).
  • a control group received only PBS.
  • FIG. 13 shows an HE-stained photograph of the excised tumor.
  • Figures 13A (x100) and B (x400) are photographs of tumor masses of A549 cells administered peptide 16
  • Figures 13C (x100) and D (x400) are controls. In the control, the tumor tissue mass was filled with cells, but in mice to which peptide 16 was administered necrosis occurred inside the tumor, suggesting that peptide 16 exhibited an antitumor effect.
  • the polypeptide of the present invention can be used for cell fusion of plant cells and animal cells.
  • the pharmaceutical composition of the present invention can be used for treating cancer.

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Abstract

La présente invention vise à fournir un procédé de fusion cellulaire efficace. L'invention concerne également un procédé permettant de tuer des cellules cancéreuses ou un procédé pour la fusion d'une enveloppe virale par fusion membranaire.　A cet effet, l'invention concerne: (1) un polypeptide, qui comprend une séquence d'acides aminés ayant un groupe représenté par la formule (I): -Z-Xm-Y (I) (dans la formule: Z est un lieur hydrophile; X est un résidu d'acide aminé hydrophile choisi dans le groupe constitué par la sérine, la thréonine, l'asparagine, la glutamine, l'arginine, l'histidine, la lysine, l'acide aspartique, l'acide glutamique, la tyrosine et la cystéine; Y est un groupe carboxyle ou un groupe amino; m est un nombre entier de 1 à 5; et, lorsque m est 2 à 5, les résidus d'acides aminés hydrophiles peuvent être identiques ou peuvent être la combinaison ou différents résidus d'acides aminés au niveau de l'extrémité N-terminale d'une séquence d'acides aminés choisie dans le groupe constitué par les séquences d'acides aminés représentées par les SEQ ID NOs : 1-8; ou (2) un polypeptide, qui comprend une séquence d'acides aminés ayant un groupe représenté par la formule (I) au niveau de l'extrémité N-terminale d'une séquence d'acides aminés dans laquelle des acides aminés 1-4 ont été supprimés, substitués, insérés et/ou ajoutés dans une séquence d'acides aminés représentée par SEQ ID NO: 1-8 et qui a une activité de fusion cellulaire ou une activité de fusion d'enveloppe virale.
PCT/JP2022/013074 2021-03-22 2022-03-22 Peptide, et agent de fusion cellulaire et composition pharmaceutique pour cancérothérapie contenant ledit peptide Ceased WO2022202785A1 (fr)

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JP2008532549A (ja) * 2005-03-15 2008-08-21 アラーガン、インコーポレイテッド 内因性クロストリジウム毒素受容体系に対する増大した標的能力を有する修飾クロストリジウム毒素
WO2011063128A1 (fr) * 2009-11-18 2011-05-26 Affinergy, Inc. Greffons osseux implantables
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WO2003099862A1 (fr) * 2002-05-24 2003-12-04 Applied Nanosystems B.V. Exportation et modification de (poly)peptides de type lantibiotique
JP2008505854A (ja) * 2004-06-18 2008-02-28 イーペーエフ ファルマシューティカルス ゲゼルシャフト ミット ベシュレンクテル ハフツング オリゴマーペプチドおよびｈｉｖ感染症の治療のためのその使用
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WO2011063128A1 (fr) * 2009-11-18 2011-05-26 Affinergy, Inc. Greffons osseux implantables
US20190284240A1 (en) * 2016-11-22 2019-09-19 Ohio State Innovation Foundation Cyclic cell penetrating peptides comprising beta-hairpin motifs and methods of making and using thereof
WO2021054448A1 (fr) * 2019-09-20 2021-03-25 美智子 甲賀 Peptide, et agent de fusion cellulaire et composition pharmaceutique pour cancérothérapie contenant ledit peptide

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WO2025225632A1 (fr) * 2024-04-23 2025-10-30 美智子 甲賀 Composition pharmaceutique pour le traitement ou la prévention de maladies infectieuses à coronavirus

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